1. Field of the Invention
The present invention concerns a method of promoting axonal regeneration. In particular, the invention concerns a method of promoting the growth or regeneration of neurons, and treating disease or conditions associated with the loss, loss of function or dysfunction of nerve cells, in particular thalamic nerve cells, by administering a polypeptide having a high degree of sequence identity with a native sequence Netrin G1 (NGL-1) or an agonist thereof.
2. Description of the Related Art
The Netrins are a family of laminin-related, diffusible axon guidance molecules that are conserved from C. elegans to vertebrates [Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. & Hedgecock, E. M. “UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans”. Neuron 9, 873-881. (1992); Serafini, T. et al. “The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6”, Cell 78, 409-424. (1994)] The vertebrate netrins are highly expressed in the ventral midline of the central nervous system (CNS), attracting the commissural axons and repelling the trochlear motor axons [Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. & Hedgecock, E. M. “UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans”. Neuron 9, 873-881. (1992)]. Axonal attraction to Netrins is mediated mainly by the transmembrane receptor DCC, whereas its repulsive action is dependent on both the DCC and Unc5 receptors [Hedgecock, E. M., Culotti, J. G. & Hall, D. H. “The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans”, Neuron 4, 61-85. (1990); Hamelin, M., Zhou, Y., Su, M. W., Scott, I. M. & Culotti, J. G. “Expression of the UNC-5 guidance receptor in the touch neurons of C. elegans steers their axons dorsally”, Nature 364, 327-330. (1993); Keino-Masu, K. et al. “Deleted in Colorectal Cancer (DCC) encodes a netrin receptor”, Cell 87, 175-185. (1996); Leonardo, E. D. et al. “Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors”, Nature 386, 833-838. (1997)].
Recently a Netrin-related molecule, Netrin-G1 (also named as Laminet-1), was identified and shown to be distinguished from the classical netrins in a number of aspects [Nakashiba, T. et al. “Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrin that is functionally divergent from classical netrins” J. Neurosci. 20, 6540-6550. (2000); Yin, Y., Miner, J. H. & Sanes, J. R. “Laminets: laminin- and netrin-related genes expressed in distinct neuronal subsets”, Mol. Cell. Neurosci. 19, 344-358. (2002)]. See also, WO 99/63088, published Dec. 9, 1999, disclosing the sequence of Netrin-G1 (originally designated PRO1133, encoded by DNA53913), and WO 01/68848, published Sep. 20, 2001, including microarray data demonstrating the over-expression of Netrin-G1 (PRO1133) in tumor.
Unlike other Netrins, Netrin-G1 is predominantly tethered to the cell membrane via a C-terminal glycosyl-phosphatidylinositol (GPI) anchor and is not expressed in the ventral midline of the CNS. Instead it is found in sets of projection neurons such as the mitral cells of the olfactory bulb, the deep cerebellar nuclei and the dorsal thalamus. Multiple splice variants of Netrin-G1 have been uncovered, suggesting potential complexity of this gene. More importantly, none of the multiple isoforms of Netrin-G1 binds DCC or Unc5, the identified netrin receptors [Nakashiba, T. et al. “Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrin that is functionally divergent from classical netrins”, J. Neurosci. 20, 6540-6550. (2000)]. Therefore its function and mode of action were not known.
The thalamocortical axons (TCAs) project from the dorsal thalamus to the cerebral cortex. These axons first extend toward the ventral thalamus and then turn 90° rostrally, coursing through the ventral telencephalon (i.e. subpallium/the striatum) within the internal capsule and they turn dorsally to reach their final target, the cerebral cortex. Along this complex trajectory, both attractive and repulsive signals guide the thalamocortical axons [Braisted, J. E., Tuttle, R. & O'Leary D, D. “Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path”, Dev. Biol. (Orlando) 208, 430-440 (1999)]. Specifically, the repulsive signals Slit-1 and Slit-2 are required to steer the thalamocortical axons away from the ventral midline region of the diencephalons [Bagri, A. et al. “Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain”, Neuron 33, 233-248. (2002)], while Netrin-1 expressed in the ventral telencephalon appear to attract the thalamocortical axons into the internal capsule [Braisted, J. E. et al. “Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection,” J. Neurosci 20, 5792-5801 (2000)]. However, a substantial population of the thalamocortical axons is still able to reach the internal capsule and the cerebral cortex in the Netrin1-deficient mice [Bagri, A. et al. “Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain.” Neuron 33, 233-248. (2002)] suggesting that additional attractive factor(s) must be involved.
A leucine-rich repeat containing polypeptide designated PRO331 (encoded by DNA40981), was disclosed in WO 99/142328, published Mar. 25, 1999 and WO 00/15796, published Mar. 23, 2000, and shown to inhibit VEGF-stimulated proliferation of endothelial cell growth. WO 01/04311, published Jan. 18, 2001 disclosed results demonstrating that the same molecule has pro-inflammatory properties.
In Example 171 of WO 00/73454, published Dec. 7, 2000, PRO331 was shown to bind PRO1133 (now named Netrin-G1) and vice versa.